Agonist-induced activation of human FFA1 receptor signals to extracellular signal-regulated kinase 1 and 2 through Gq- and Gi-coupled signaling cascades

Qian et al. Cellular & Molecular Biology Letters (2017) 22:13 DOI 10.1186/s11658-017-0043-3 RESEARCH Cellular & Molecular Biology Letters Open Access Agonist-induced activation of human FFA1 receptor signals to extracellular signalregulated kinase 1 and 2 through Gq- and Gi-coupled signaling cascades Jing Qian1* , Yuyang Gu1, Chun Wu2, Feng Yu1, Yuqi Chen1, Jingmei Zhu1, Xingyi Yao1, Chen Bei1 and Qingqing Zhu1 * Correspondence: 1 Huzhou University Schools of Nursing and Medicine, Huzhou University, HuZhou 313000, China Full list of author information is available at the end of the article Abstract Background: FFA1 is abundantly expressed in the liver, skeletal muscle, monocytes and nervous system, but is particularly abundant in pancreatic β cells. It is widely believed that FFA1 exerts its regulatory roles in a variety of physiological and pathological functions. In response to oleic acid, FFA1 has been shown to induce the activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) through a mechanism involving EGFR transactivation in a breast cancer cell line. However, the underlying molecular mechanism for ERK1/2 activation mediated by n-6 free fatty acid (LA) in HEK293 cells remains to be further elucidated. Methods: A FLAG-FFA1 vector was stably expressed in HEK293 cells. Western blot analysis was applied to investigate the change in LA-induced ERK1/2 phosphorylation change in response to kinase inhibitors. Arrestin-2/3-specific siRNA was used to analyze the effect of arrestin-2/3 knockdown on FFA1-mediated ERK1/2 activation. Results: We proved that activation of ERK1/2 by LA was rapid, peaking at 5 min. Further experiments proved that FFA1 couples to a Gq protein and activates PI-PLC, which induces the IP3/Ca2+ and DAG/PKC signal pathways, both of which are involved in ERK1/2 activation. We also showed that there is no EGFR transactivation, arrestin-2/3 or Gβγ pathway participation in ERK1/2 phosphorylation. Treating cells with PTX abolished ERK1/2 activation at a late time point (≥20 min), indicating a critical role for Gi subunits in FFA1-mediated ERK1/2 activation. Conclusions: Our study provides a detailed delineation of the LA-mediated activation of ERK1/2 in HEK293 cells that are stably transfected with human FFA1. We also present evidence of Gi/Gq-induced synergism in the regulation of ERK1/2 phosphorylation. These observations may provide new insights into the pharmacological effects of FFA1 and the physiological functions modulated by FFA1-mediated activation of ERK1/2. Keywords: FFA1, Phosphatidylinositol-specific phospholipase C, Extracellular signalregulated kinase 1 and 2, Gαq/11, Gαi/o Background FFA1 was sequenced and identified as a member of a subfamily of intronless GPCRs. The family includes GPR41, GPR42 and GPR43, all of which reside on chromosome © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Qian et al. Cellular & Molecular Biology Letters (2017) 22:13 19q13.1. The receptor encoded by FFA1 contains two N glycosylation sites (N-X-S/T), five protein kinase C (PKC) phosphorylation sites, and a cysteine at the C-terminus [1]. Both saturated and unsaturated medium- and long-chain (C12-C22) free fatty acids (FFA) were identified as endogenous ligands for FFA1 [2]. FFA1 is abundantly expressed in rodent insulin-secreting cell lines, including INS-1E cells [3], Min6 cells [4] and pancreatic β cells [2]. It is also found in the human brain [5]. Accumulating evidence shows that FFA1 plays a crucial role in the regulation of glucose homeostasis mediated by free fatty acid-induced insulin secretion [6, 7]. In addition to the regulation of endocrine function, FFA1 is involved in bone remodeling, inflammation and neurogenesis [8–10]. These findings suggest that FFA1 may work through multiple pathways in the regulation of different physiological functions. The basic understanding of G-protein-coupled receptors (GPCRs) is that almost all signal through extracellular signal-regulated kinase (ERK) signaling cascades, which are associated with about 200 cellular substrates and mediate a variety of cellular processes, including proliferation, differentiation, migration, survival and apoptosis [11–13]. FFA1 is activated to elicit an increase in intracellular Ca2+ levels via the Gqdependent pathway, leading to enhancement of glucose-stimulated insulin secretion [14, 15]. In addition, FFA1 has been shown to induce ERK1/2 activation through a mechanism involving Src kinase and EGFR transactivation by oleic acid in the breast cancer cell lines MCF-7 and MDA-MB-231 [16]. By contrast, another study revealed that FFA1 activation by n-3 fatty acids can abolish EGF-induced proliferation and migration in MCF-7 and MDA-MB-231 cells [17]. This suggests that the effects of different FFA1 agonists are different. In mouse embryonic stem cells, LA regulates various cell cycle proteins via p44/42 MAPK signaling [18]. Interestingly, unsaturated fatty acids promote the activation of ERK1/2 mainly via FFA1, leading to an anti-lipoapoptotic effect on NIT-1 cells [19]. Thus, the underlying mechanism regulating ERK1/2 activation mediated by the n-6 free fatty acids (LA) in HEK293 cells through FFA1 remains unclear. It is now known that GPCRs regulate MAPK cascades via distinct Gi-, Gs-, Gq/11- and Gβγ- dependent signaling pathways, leading to activation of ERK1/2 [20]. G proteins have complex and diverse roles in the FFA1 signal pathway. Pharmacological inhibitionof Gq/ 11 blocked Ca2+ release from the ER in the β cell line INS-1E [3]. In HEK cells, TUG424mediating FFA1 downstream signaling is inhibited by pertussis toxin (PTX), which indicates that Gi/o is partially involved [21]. Moreover, LA have been proven to decrease the voltage-gated K+ current through FFA1/Gs/cAMP/protein kinase A (PKA) [22]. Further exploration of G protein subunits in FFA1-mediated ERK1/2 activation will be important to better understand the role of FFA1 in various physiological functions. Methods Materials Lipofectamine 2000, G418 and Opti-MEM were purchased from Invitrogen. Cell culture media and fetal bovine serum were obtained from Hyclone. Pertussis toxin (PTX), Go6983 and thapsigargin were purchased from Sigma. UBO-QIC was purchased from Dr. E. Kostenis at the University of Bonn. U0126, tyrphostin AG1478, GM6001, PP2 and ET-18-och3 were from Calbiochem. Monoclonal anti-FLAG antibody was Page 2 of 12 Qian et al. Cellular & Mo (...truncated)